Suppression of antimicrobial protein levels

ABSTRACT

Exercise is a stressor that is known to in some cases suppress antimicrobial protein levels, particularly secretory immunoglobulin type A (IgA). Athletes in the midst of in-season training often demonstrate low levels of sIgA and supplements may enhance post exercise mucosal immune function. A study was conducted where three treatments were administered (botanical spray, botanical drops, placebo) at the onset of 30 minutes of 80% V02 max cycle ergometer exercise. Secretory IgA and Human Alpha Defensin were quantified in saliva samples 30 minutes and 90 minutes post exercise. Analysis revealed a significant treatment effect at 30 min (p=0.030) with post hoc testing revealing a difference between the botanical spray and placebo (p=0.027), but by 90 minutes there was no differences by treatment (p=0.758). Based upon this study, it appears that a single dose of the tested botanical can provide enhanced mucosal immune capability for a short time post exercise.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.16/242,095 titled “SUPPRESSION OF ANTIMICROBIAL PROTEIN LEVELS” FILEDJan. 8, 2019, which claims benefit to a U.S. Provisional Application No.62/614,522 titled “SUPPRESSION OF ANTIMICROBIAL PROTEIN LEVELS” filed onJan. 9, 2018.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

REFERENCE TO A “SEQUENCE LISTING”, A TABLE, OR COMPUTER PROGRAM

Table 1 provides a statistics analysis describing physicalcharacteristics of participates of the study.

Table 2 presents the measure concentration of salivary IgA before andafter exercise.

Table 3 presents an analysis of variance for measurements corrected forosmolality.

Table 4 presents an analysis of variance for measurements controlled forchanges in total protein.

Table 5 presents an analysis for measurement controlled for changes insalivary flow rate.

Table 6 presents an analysis of variance for salivary alpha amylaseactivity.

Table 7 presents an analysis of variance for proline rich proteinconcentration.

FIELD OF THE INVENTION

The present invention relates to the general field of kinesiology,especially as it relates to prevention of illness in athletes. Theinvention relates generally to a system and method of administratingbotanicals. In particular, the invention relates to administration ofBiocidin®.

BACKGROUND OF THE INVENTION

Exercise is a known stressor that has influence over many differentphysiological systems in the body. There is evidence in the literaturethat long-term training in athletes can negatively affect antimicrobialprotein levels in the oral cavity. Furthermore it has been reported thatathletes face a higher incidence of upper respiratory tract infection(URTI) compared to more sedentary individuals. Based upon the reports inthe literature, it is understood that long-term training for sportperformance is a stressor that can both elevate physiological potentialfor in the athletic arena, but may depress function in other system.Moreira et al. reported that a 2-week detraining period after acompetitive soccer season attenuated Immunoglobulin A (sIgA) suppressionand symptomology of URTI due to training. From this study it can besuggested that mucosal immunity can quickly rebound if rest and recoveryare allowed for athletic populations. However, the demands placed uponathletes, reduced mucosal immunity and risk for URTI are likely toremain an issue into the future.

It has also been determined by previous research that certain forms ofacute athletic performance and exercise have the ability to suppressimmunoglobulin secretion in the oral cavity. However, most of theseexercises are long duration and aerobic, the evidence for the changes inantimicrobial protein with short-term intermittent exercise is lessclear with some evidence suggesting no change in immunoglobulin type A.Additionally, Li and Gleeson reported that 60% maximum aerobic capacity(V02 max) cycling for 2 hours did not negatively impact sIgA secretionrates. However, MacKinnon and Jenkins had previously reported a declinein sIgA with intense interval exercise (0.075 g*kg⁻¹) on a cycleergometer. Though there is much study yet needed to fully elucidate theresponses of antimicrobial proteins in the saliva with all forms ofexercise there is enough evidence that further study is warrantedparticularly regarding methods to increase antimicrobial protein levels.

Some botanicals, such as baker's yeast beta glucan, have been shown toincrease sIgA in saliva and help decrease symptoms of cold and flu postexercise. Shiitake mushrooms are another natural food substance that isknown to contain beta glucans, which may be beneficial for human health.Shiitake mushrooms have been demonstrated to have anti-oxidant activitypost exercise, but no evidence currently exists to examine changes inoral anti-microbial protein levels post exercise. Therefore, the purposeof the initial investigation was to examine the effects of a botanicalsupplement on secretory immunoglobulin A and alpha defensing levelspost-exercise.

DETAILED DESCRIPTION OF THE INVENTION

A series of studies were conducted to evaluate the effectiveness ofBiocidin®. Biocidin® is comprised of Bilberry extract (25%anthocyanosides), Noni, Milk Thistle, Echinacea (purpurea &angustifolia), Goldenseal, Shiitake, White Willow (bark), Garlic,Grapeseed extract (min. 90% polyphenols), Black Walnut (hull and leaf),Raspberry, Fumitory, Gentian, Tea Tree oil, Galbanum oil, Lavender oil(plant & flower), and Oregano oil (plant & flower). For the study 20apparently healthy male subjects were recruited to participate.Measurements and an associated statistics analysis describingparticipates is presented in Table 1. On the first visit the subjectsgave informed consent after being briefed on the study procedures andthen had basic anthropometric information collected (height, weight,body fat percentage determined via 3 site skinfold analysis). Thesubjects also practiced providing a passive drool sample according tothe methods laid out in the Standard Operating Procedures for the HumanPerformance Lab at the University of Louisiana at Lafayette. Followingthis assessment the subjects had a tests of maximum aerobic capacity(VO2 max) administered on a cycle ergometer with inspired gasesmonitored with a CosMed Cardio Pulmonary Exercise Test. In brief, thistest consisted of pedaling at a cadence of 60-80 rpm on anelectronically braked “bicycle” on which the workload was increasedevery 2 minutes until the participate voluntarily stops exercising ortheir oxygen consumption has ceased increasing as determined by computerdata assessed in real time. This test concluded the activities of thefirst lab visit.

The next 2 lab visits were conducted in the morning. The participantsreported to the lab in an overnight fasted state without having brushedtheir teeth. The exercise for these visits involved a 30 minute cyclingprotocol at 70% of the maximum workload (in watts) achieved during thetest of maximum aerobic capacity. The participants provided a baseline,30 min and 90 min post exercise saliva samples (2 ml total volume percollection, 5 minute timed collection) into pre-weighted cryovials.Prior to each exercise session the participants received one of twotreatments in a random order. The treatments consist of saline solutionto be sprayed in the oral cavity (small manual squirt bottle), orBiocidin solution in a spray bottle. The participants received a seconddose immediately after exercise and after the 30 minute saliva samplewere collected.

After collection the saliva samples were frozen at −35 degree Celsius ina dedicated freezer in a secure laboratory with limited access. Prior toanalysis the saliva samples was allowed to come to room temperature andcentrifuged at 1500 g for 15 minutes.

Saliva samples were tested for osmolality via a vapor pressure osmometer(EliTech VAPRO). The instrument has a measurement range of 20 to 3200mmol*kg and requires 10 pg of samples. The Osmometer was calibratedprior to testing using a three point linear calibration. The salivasamples were tested immediately after collection, prior to being frozenfor storage.

Saliva samples were tested for total protein content after thawing. Theanalysis was conducted on a Qubit 3.0 Fluorometer using a standard totalprotein assay kit. (Fisher Scientific, Qubit Protein Assay kit). Theassay kit has a range of 12.5 pg/ml to 5 mg/ml with 1iAL to 204 ofsample.

After collection, saliva samples were weighted on an analytic balance(Ohaus, Model PA313) to determine the salivary flow rates. These finalweights were subtracted from the weight of the vial. A density of 1.0g/ml was assumed and the final weights were divided by 5 min (time ofcollection) to arrive at ml/min secretion rate.

Thawed saliva samples were analyze for salivary alpha amylase activity(SAA) using a commercial calorimetric assay (Salimetrics, Inc). Theassay involves the detection of 2-chloro-p-nitrophenol separated frommaltotriose which can be detected at 405 nm. The assay was conducted instandard microplates and read with a kinetic microplate reader (BioTekELx 808uv).

Thawed saliva samples were analyzed for Salivary Immunoglobulin A induplicate using a commercial sandwich ELISA (Eagle Biosciences). Theabsorbance was read at 450 nm with a reference filter at 690 nm.

Salivary Praline Rich Protein and Bacterial Permeability IncreasingProtein biomarkers were analyzed using sandwich ELISA kits (Cloud-CloneCorp.). Analysis was conducted in duplicate and read at 450 nm.

Changes in biomarkers by treatment (Biocidin® vs Placebo) by time (pre,post 30, post 90) were assessed via Repeated Measures analysis ofvariance (ANOVA). Post Hoc Analysis was carried out via pairedsamplest-tests for all significant interaction effects. All statisticalprocedures were carried out using a modern statistical software packageOMP 12.0 Pro). Statistical significance was set a priori at alpha <0.05.

With relatively few exceptions Bacterial Permeability Increasing Protein(BPI) was not detected in the saliva collected with ELISA, and thereforecould not be statistically analyzed. The assay kit has a limit ofdetection of 31 pg/ml, which is sufficient to detect BPI in theconcentrations listed in some publications. However, the actual normalconcentrations of BPI in human saliva are not well characterized. Untila greater understanding of the normal concentrations of BPI in salivacan be developed including any other potential confounders (such asdiurnal effects) this biomarker should not be included in furtherstudies.

Salivary IgA data was examined in 4 different manners: 1) measuredconcentration (see Table 2), 2) controlled for changes in Osmolality(see Table 3), 3) controlled for changes in total protein (see Table 4)and 4) controlled for changes in Salivary Flow Rate (see Table 5).Repeated measures ANOVA for concentrations (raw) did not reveal a maineffect for treatment (F=0.008, p=0.929) nor a significant effect fortreatment by time (F=1.03, p=0.653)

As presented in Table 3, repeated measures ANOVA for concentrations(controlled for Osmolality) did not reveal a main effect for treatment(F=0.233, p=0.636) nor a significant effect for treatment by time(F=0.561, p=0.607)

As presented in Table 4, repeated measures ANOVA for concentrations(controlled for total protein) did not reveal a main effect fortreatment (F=0.118, p=0.733) but did reveal a significant effect fortreatment by time (F=2.60, p=0.044). Post hoc testing did reveal asignificant different at the 90 minute post exercise time point(p=0.05).

As presented in Table 5, repeated measures ANOVA for concentrations(controlled for salivary flow rate) did not reveal a main effect fortreatment (F=0.020, p=0.889) nor a significant effect for treatment bytime (F=2.05, p=0.071)

As presented in Table 6, repeated measures ANOVA for Salivary AlphaAmylase activity did not reveal a main effect for treatment (F=0.007,p=0.934) nor a significant effect for treatment by time (F=0.315,p=0.731)

As presented in Table 7, repeated measures ANOVA for Proline RichProtein (PRP) concentrations did not reveal a main effect for treatment(F=0.0452, p=0.832) nor a significant effect for treatment by time(F=0.901, p=0.419)

The results of the present study do lend additional support to theefficacy of Biocidin® spray for post exercise mucosal immunity support.While biomarkers such as SAA, PRP and BPI were not increased withBiocidin®, the change noted for sIgA is highly relevant as this is themost abundant antimicrobial protein (AMP) present in saliva. It is alsoimportant to note that controlling the analysis of sIgA for totalprotein content has been previously reported on in the literature inresponse to exercise stress (MacKinnon and Jenkins, 1993). More commonlystudies control sIgA for salivary flow rate through the extrapolation ofa secretion rate (Akimoto, 2003). It should be noted that whilenon-significant a trend in the data for sIgA secretion rate that wassimilar to the finding for sIgA/total protein was noted. These paralleldata lend support to the conclusion that Biocidin® is indeed effectivefor augmenting sIgA post exercise.

The lack of findings from the BPI and PRP may be due to the lack ofavailable data regarding the response of these AMPs to exercise. It ispossible that these are not responsive to exercise stimuli. SalivaryAlpha Amylase was notably lower during the pre-exercise time, a findingthat has been previously reported (Walsh et al., 1999) and is likely dueto anticipatory psychological stress. This biomarker is also know toincrease post exercise, which was noted in the results of the presentstudy. However, nutritional supplements that are effective at augmentingslga such as beta glucan (McFarlin et al., 2013), have not beeninvestigated with Salivary alpha amylase. Given the responsiveness ofsalivary alpha amylase to sympathetic tone (Messina et al. 2016) the preexercise anticipatory phase and increased sympathetic activity duringexercise may mask other potential changes in activity level.

TABLE 1 Participants (M ± SD) Height (cm) 176.4 ± 4.7  Weight (kg)  82.8± 13.3 Body Fat (%) 14.7 ± 4.3 V02 max (ml/kg*min) 27.4 ± 4.8

TABLE 2 Treatment (M ± SD) Pre Exercise 30 min Post Ex 90 min Post ExBiocidin (μg/m1) 296.9 ± 347.6 246.0 ± 261.4 294.8 ± 394.4 Placebo(μg/m1) 279.1 ± 276.9 305.9 ± 362.7 276.7 ± 405.9

TABLE 3 Treatment (M ± SD) Pre Exercise 30 min Post Ex 90 min Post ExBiocidin 4.85 ± 5.98 4.27 ± 5.06 4.77 ± 6.62 (pg/mrmOSM/kg¹) Placebo4.71 ± 5.15 5.93 ± 8.12 5.89 ± 9.22 (μg/ml*mOSM/kg⁻¹)

TABLE 4 Treatment (M ± SD) Pre Exercise 30 min Post Ex 90 min Post ExBiocidin .886 ± 1.31 .630 ± .703 .928 ± 1.31* (gg/ml*ng protein⁻¹)Placebo .737 ± .704 .778 ± .902 .667 ± .868  (μg/ml*pg protein⁻¹)*denotes significantly different than placebo

TABLE 5 Treatment (M ± SD) Pre Exercise 30 min Post Ex 90 min Post ExBiocidin (ig/min) 61.8 ± 97.6  38.8 ± 53.1 65.9 ± 94.24 Placebo (μg/min)54.6 ± 63.67 62.4 ± 63.2 57.7 ± 93.2 

TABLE 6 Treatment (M ± SD) Pre Exercise 30 min Post Ex 90 min Post ExBiocidin U/ml 34.3 ± 32.4 68.1 ± 128.5 37.8 ± 41.45 Placebo U/ml 39.7 ±48.7 51.2 ± 57.1  45.6 ± 58.5 

TABLE 7 Treatment (M ± SD) Pre Exercise 30 min Post Ex 90 min Post ExBiocidin  842.2 ± 1313.6 1095.92 ± 1719.9  951.65 ± 1448.7 (ng/ml)Placebo 1108.5 ± 1517.8  856.52 ± 1388.7 1279.61 ± 1671.9 (ng/ml)

We claim:
 1. A method for suppressing upper respiratory tract infection comprising administrating a dose of biocidin.
 2. The method of claim 1, wherein the dose of biocidin is administered prior to exercise.
 3. The method of claim 1, wherein the dose of biocidin is administered during exercise.
 4. The method of claim 1, wherein the dose of biocidin is administered after exercise.
 5. The method of claim 1, wherein the dose of biocidin is administered by spraying biocidin in the oral cavity. 